Volume 63, Issue 4, Pages 1240-1248 (April 2003) Cux-1 transgenic mice develop glomerulosclerosis and interstitial fibrosis  Jennifer G. Brantley, Madhulika Sharma, Neal I. Alcalay, Gregory B. Vanden Heuvel  Kidney International  Volume 63, Issue 4, Pages 1240-1248 (April 2003) DOI: 10.1046/j.1523-1755.2003.00889.x Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 1 Expression of Cux-1 protein in adult transgenic kidney. Light micrographs of wild-type (A and C) and transgenic (B and D) adult kidneys. High levels of Cux-1 protein are detected in the nuclei of glomeruli (g) and tubules of adult transgenic mice (A and B). Cux-1 is minimally expressed in glomeruli (g) or tubules of age-matched wild-type kidneys (A). Higher magnification reveals high levels of nuclear Cux-1 expression in a subset of cells in transgenic glomeruli, while Cux-1 is minimally expressed in the glomeruli of nontransgenic mice (C and D). Double labeling of transgenic kidney sections with Cux-1, in green, and desmin, in red, (E) reveals that Cux-1 is ectopically expressed in mesangial cells. Inset in (E) shows nuclear localization of Cux-1 in a cell labeled for desmin. Original magnification, (A) and (B) ×200; (C), (D), (E) ×400; inset to (E) ×630. Kidney International 2003 63, 1240-1248DOI: (10.1046/j.1523-1755.2003.00889.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 3 Increased proliferation of mesangial cells in transgenic kidneys. (A) Wild-type glomerulus. (B) Transgenic glomerulus. Proliferating cellnuclear antigen (PCNA) staining (red) is observed in transgenic glomeruli, but not wild-type glomeruli. Desmin staining (green) reveals that many of the proliferating cells are mesangial cells. Original magnification, ×630. Kidney International 2003 63, 1240-1248DOI: (10.1046/j.1523-1755.2003.00889.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 4 Mesangial increase in transgenic kidneys. Periodic acid-Schiff (PAS)–stained sections of transgenic (A and B) and wild-type (C and D) kidneys demonstrating matrix expansion in cytomegalovirus (CMV)/Cux-1 mice. The first 100 glomeruli from wild-type and transgenic kidney sections were scored for the percent PAS-positive material in each glomerulus (see Table 2). Representative glomeruli were scored as follows: (A) 51% to 75% PAS positive; (B) 26% to 50% positive; (C) and (D) 0% to 25% PAS positive. Original magnification, ×1000. Kidney International 2003 63, 1240-1248DOI: (10.1046/j.1523-1755.2003.00889.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 5 Localization of type IV collagen in transgenic glomeruli. (A) Wild type glomerulus. (B) Transgenic glomerulus. Type IV collagen staining in the mesangial matrix is increased in transgenic glomeruli. Original magnification, ×400. Kidney International 2003 63, 1240-1248DOI: (10.1046/j.1523-1755.2003.00889.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 6 Interstitial fibrosis in Cux-1 transgenic kidneys. Masson-trichrome staining of wild-type (A and B) and transgenic (C and D) kidney sections showing interstitial fibrosis in peritubular spaces of transgenic kidneys. Original magnification, (A) and (C) ×50; (B) ×100; (D) ×200. Kidney International 2003 63, 1240-1248DOI: (10.1046/j.1523-1755.2003.00889.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 7 Western blot of type IV collagen. Fifty micrograms of total kidney lysate isolated from wild-type and transgenic 6-month-old kidneys was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The arrow indicates an increase in type IV collagen in transgenic kidneys. Positions of molecular weight standards (×kD) are shown on right. Kidney International 2003 63, 1240-1248DOI: (10.1046/j.1523-1755.2003.00889.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 8 Albuminuria in Cux-1 transgenic mice. Two and six microliters of urine from wild-type (lanes 1 and 2) and transgenic (lanes 3 and 4) mice, respectively, was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The presence of albumin in the urine was detected using a sheep polyclonal antibody to albumin. Twenty micrograms of bovine serum albumin (BSA) was added as a control. Kidney International 2003 63, 1240-1248DOI: (10.1046/j.1523-1755.2003.00889.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 9 Electron micrographs of glomeruli. Wild-type (A) and transgenic (B) glomeruli are shown at medium (×14,400) magnification. The glomerular basement membrane appears normal, but many foot processes (FP) are fused (FFP) in transgenic mice. L is capillary lumen. Kidney International 2003 63, 1240-1248DOI: (10.1046/j.1523-1755.2003.00889.x) Copyright © 2003 International Society of Nephrology Terms and Conditions